骨形态发生蛋白-2对人脐带间充质干细胞增殖和分化的影响  

Effect of bone morphogenetic protein-2 on proliferation and osteogenic differentiation of human umbilical cord mesenchymal stem cells in vitro

作　　者：杨大志[1] 贺永进[1] 郭晓东[1] 郑启新[1] 

机构地区：[1]华中科技大学同济医学院附属坼和医院骨科,武汉430022

出　　处：《中华实验外科杂志》2010年第11期1602-1605,共4页Chinese Journal of Experimental Surgery

基　　金：“十一五”国家高技术研究发展计划（863）干细胞与组织工程重大项目（2006AA02A124863）;国家自然科学基金资助项目（30973027）;教育部“新世纪优秀人才支持计划”资助项目（NCET-05-0647）

摘　　要：目的 观察骨形态发生蛋白-2（BMP-2）对人脐带间充质干细胞（hUCMSCs）增殖和分化的影响.方法 体外培养hUCMSCs,在培养基中加入20 mg/L BMP-2,噻唑蓝（MTT）比色法观察BMP-2对hUCMSCs的增殖效果,流式细胞术检测BMP-2作用后细胞表面STRO-1的表达,逆转录-聚合酶链反应（RT-PCR）测量BMP-2作用后hUCMSCs骨桥蛋白（OPN）、碱性磷酸酶（ALP）、Ⅰ型胶原蛋白（COL1）的mRNA表达变化,碱性磷酸酶染色观察hUCMSCs在BMP-2培养基作用下ALP染色变化,Von kossa染色实验观察BMP-2对hUCMSCs钙结节的形成.结果 细胞在未加BMP-2和加BMP-2培养基中培养1、3、5、7 d,虽然细胞的增殖率上升,但各组间比较差异无统计学意义（P＞0.05）,同时发现在2%血清的培养基中培养7 d后,hUCMSCs的增殖促进约10%左右.BMP-2培养7 d后细胞表面STRO-1阳性细胞比例上升明显,由25.1±4.0上升至51.1±6.4,差异有统计学意义（P＜0.01）.BMP-2培养条件下COL1 mRNA的表达增强,差异有统计学意义（P＜0.05）,OPN mRNA出现表达,ALP mRNA比无BMP-2培养明显增强,差异有统计学意义（P＜0.05）.在BMP-2培养条件下ALP染色出现大片细胞阳性染色.在培养28 d,Von kossa染色出现明显的钙结节.结论 BMP-2对hUCMSCs成骨诱导分化作用明显,而增殖作用很弱.Objective To study the effects of the bone morphogenetic protein-2 （BMP-2） on proliferation and osteogenic differentiation of human umbilical cord mesenchymal stem cells （hUCMSCs） in vitro. Methods hUCMSCs were cultured in vitro, and BMP-2 （20 mg/L） was added into the culture medium. The proliferation effect of BMP-2 on hUCMSCs was examined by MTT assay. The proportion of antigen-positive cells for STRO-1 was observed by flow cytometry （FCM）. The osteopontin （ OPN）, alkaline phosphatase （ALP） and type 1 collagen （ COL1 ） mRNA levels was detected by reverse transcription-polymerase chain reaction （ RT-PCR）. The ALP activity was tested by ALP staining array. Von Kossa staining method was used to study the calcification effects. Results The proliferation rate was increased 1-, 3-,5-, 7-day BMP-2 （20 mg/L） groups and no BMP-2 group, but there was no statistically significant difference among groups （P 〉 0. 05）. When the concentration of serum in the culture medium decreased from 10% to 2%, the proliferation was just increasing 10% and no different between BMP-2 group and no BMP-2 group （P〉0.05）. The percentages of STRO-1 positive cells was increasing from 25.1 ±4.0 to 51.1 ±6.4 after culture with BMP-2（ P 〈 0.01 ）. The BMP-2 increased the ALP and COL1 gene expression, induced the OPN gene expression. A lot of hUCMSCs was positive in ALP staining array in BMP-2 medium. Many mineralized nodes was detected in the Von kossa staining method after culture 28 days in the BMP-2 medium.Conclusion BMP-2 can promote the osteogenic differentiation,and have a little effect on the prolferation of hUCMSCs.

关 键 词：骨形态发生蛋白-2 脐带间充质干细胞 成骨分化 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]